A Turn-on Fluoresence Sensor for Hg2+ in Food Based on FRET

Subscriber access provided by NORTH CAROLINA A&T UNIV

Food Safety and Toxicology 2+

A Turn-on Fluoresence Sensor for Hg in Food Based on FRET Between Aptamers Functionalized Upconversion Nanoparticles and Gold Nanoparticles Yan Liu, Qin Ouyang, Huanhuan Li, Min Chen, Zheng-Zhu Zhang, and Quansheng Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00546 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

Journal of Agricultural and Food Chemistry

1

A Turn-on Fluoresence Sensor for Hg2+ in Food Based on

2

FRET

3

Nanoparticles and Gold Nanoparticles

4

Yan Liu a, Qin Ouyang a, Huanhuan Li a, Min Chen a, Zhengzhu Zhang b, Quansheng

5

Chen a,b,∗

Between

Aptamers

Functionalized

Upconversion

6

a

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China

7

b

State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 210036, China

∗ Corresponding author. Tel.: +86-511-88790318. Fax: +86-511-88780201. E-mail: [email protected] (Q.S.Chen)

ACS Paragon Plus Environment


8

ABSTRACT

9

In this study, a turn-on nanosensor for detecting Hg2+ was developed based on the fluorescence

10

resonance energy transfer (FRET) between long-strand aptamers functionalized upconversion

11

nanoparticles (UCNPs) and short-strand aptamers functionalized gold nanoparticles (GNPs). In the

12

absence of Hg2+, FRET between UCNPs and GNPs occurred owing to the specific matching between

13

two aptamers, result in the fluorescence quenching of UCNPs. In the presence of Hg2+, long-stranded

14

aptamers foldback into a hairpin structure due to the stable binding interactions between Hg2+ and

15

thymine, leading to the release of GNPs from UCNPs, result in the quenched fluorescence restoration.

16

Under the optimized conditions, the nanosensor achieved a linear detection range of 0.2 - 20 µM and

17

a low detection limit (LOD) of 60 nM. Meanwhile, it showed good selectivity and has been applied

18

to detecting Hg2+ in tap water and milk samples with good precision.

19

KEYWORDS: Upconversion nanoparticles; Gold nanoparticle; Aptamers; Fluorescence resonance

20

energy transfer; Turn-on fluoresence sensor; Hg2+


Page 2 of 29

Page 3 of 29

21


INTRODUCTION

22

Mercury ions (Hg2+), as common environmental pollutants, can enter human body through food

23

chains and harm the health even at low concentration 1. Due to its toxic effects, many countries have

24

developed strict restriction on the level of mercury residue in food, of which the lowest limitation

25

standards limit of mercury is 0.02 mg/kg (ca. 10 µM). For satisfying the mercury limitation

26

requirements, develop more sensitive detection method featured with simplicity and selectivity will

27

become an urgent mission in food monitoring. To satisfied the low concentration detecting, several

28

methods with intuitive results and simple operation for detecting Hg2+ have been proposed in recent

29

years, such as colorimetric assays

30

spectroscopy

31

analytical method has become core keywords, with advantages of high sensitivity, low detection

32

limit and high analytical precision. To date, various fluorescent chemosensors have been developed

33

by generally using rhodamine

34

downconversion fluorophores

35

synthesis of these fluorophores and their poor water-solubility limited their practical application in

36

food detection. Besides, conventional down-conversion emitting properties make them usually suffer

37

from high background fluorescence of endogenous chromophores in samples. Therefore, developing

38

a new simple sensor with low background and high sensitivity for Hg2+ detection is still a valuable

39

challenge.

2, 3

14-18

, and surfance enhanced Raman scattering (SERS)

4-6

, benzothiazole derivatives

11-13

4-13

, fluorescent analytical method

, plasmon resonance

19-21

. In particular, fluorescent

7-10

, and other chemical synthesized

as fluorescent probes. However, the difficult and danger in the

40

Comparing with the conventional fluorescent materials, such as organic molecules, C-dots and

41

quantum dots (QDs), upconversion nanoparticles (UCNPs) possess many outstanding features such

42

as displaying high Stokes shift, showing high resistance to photobleaching, providing continuous ACS Paragon Plus Environment


43

luminescence, inducing only a very weak autofluorescence background, avoiding photo degradation

44

in samples and enhancing signal-to-noise ratio22. More importantly, lanthanide-doped UCNPs can

45

convert longer wavelength light to shorter wavelength light through a photon upconversion process

46

by doping different ratios of rare earth ions23-25, further preventing background autofluorescence.

47

Meanwhile, Easy tunability of emission wavelength and high sensitivity also extends the application

48

field of the UCNPs. Among the application in Hg2+ detection, most are based and widespread

49

applicability in FRET systems due to their high extinction coefficients, easy synthesis and surface

50

chemistry modification. However, almost all of these biosensor are based on the mechanism of

51

fluorescence quenching induced by FRET. Compared to these fluorescence quenching probes,

52

fluoresence turn-on sensors are more suitable for practical applications, because they can effectively

53

avoid the wrong response and have a higher signal-to-noise in dark background26, 27.

54

Aptamers, are single-stranded DNA or RNA sequences, which can be isolated through systematic

55

evolution of ligands by exponential enrichment (SELET) in vitro. They have functions similar to

56

antibodies but have yet another merits of lower cost, easily synthesized and modified, and good

57

stability. In addition, aptamers are able to bind with target molecules with high selectivity and

58

specificity, so they have been widely used as recognition elements in biosensors. However, most

59

biosensor based on aptamer were used to sensing bacteria, antibiotics, DNA and other molecules,

60

few were sensing metal ion. Although the aptamer for Hg2+ have been designed based on the stable

61

binding interactions between Hg2+ and thymine, there were no turn-on FRET systems that have been

62

explored for Hg2+ detection.

63

Thus, taking advantage of the unique properties of UCNPs, GNPs and aptamers, herein, we report

64

a turn-on upconversion fluorescence biosensor for Hg2+ detection sensitively. Long-stranded aptamer ACS Paragon Plus Environment

Page 4 of 29

Page 5 of 29


65

modified NaYF4:Tm3+, Yb3+ UCNPs serve as fluorescences donors, while the complementary

66

short-stranded modified GNPs were employed as quenchers. Due to the complementary pairing of

67

the short-stranded aptamer and long-stranded aptamer, the distance between UCNPs and GNPs was

68

shortening, the FRET occurred because of spectral overlap between UCNPs fluorescence emission

69

and GNPs absorption, leading to fluorecence quenching of UCNPs. Thus, the fluorescence signal of

70

detection system was weak in the initial state. When Hg2+ was introduced, rich T-T mispairs in

71

long-strand aptamer can selectively capture Hg2+ ions and form T-Hg2+-T base pairs, causing

72

long-stranded aptamer folded back, a phenomenon which leads to the separate of GNPs from UCNPs,

73

make the FRET diminish, thereby result in the recovery of fluorescence. On these bases, the propose

74

sensor can realize sensitive and selective testing of Hg2+ with LOD of 60 nM. In addition, it is able to

75

sense Hg2+ in food samples.

76

2. EXPERIMENTAL SECTION

77

2.1 Materials

78

Rare-earth chloride hexahydrate: Ytterbium(III) chlorid hexahydrate (99.9%), yttrium (III) chlorid

79

hexahydrate (99.9%), holmium (III) chlorid hexahydrate (99.9%), oleic acid (OA, 90 %),

80

3-Aminopropyltriethoxysilane (APTES), 1-octadecene (ODE, 90 %), tetraethoxysilane (TEOS) were

81

obtained from Sigma-Aldrich. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

82

(EDC), N-Hydroxysuccinimide (NHS), succinic anhydride, hydrogen tetrachloroaurate hydrate

83

(HAuCl4, 99.9%) were supplied by Macklin reagent Ltd. (Shanghai, China). All aptamers were

84

supplied by Sangon Biotechnology Ltd. (Shanghai, China), the sequences of aptamers are as follows:

85

(long-stranded aptamer) 5' NH2 C6-CTA CAG TTT CAC CTT TTC CCC CGT TTT GGT GTT T-3',



86

(short-stranded aptamer) 5' SH C6-GAA ACT GTA G-3'. Methanol, ethanol, cyclohexane, sodium

87

hydroxide, ammonium fluoride, ammonia (25%), methylbenzene and trisodium citrateand were

88

standard analytical pure and provided by Sinopharm chemical reagent Co.,Ltd. (Shanghai, China).

89

2.2 Instruments and characterizations

90

The nanoparticles size and morphology were observed on a JEM-2100 (HR) transmission electron

91

microscopy (Japan Electron Optics Laboratory, Japan) at 120 kV acceleration voltage by placing a

92

drop of sample solution on a TEM copper grid. Powder X-ray diffraction (XRD) were determined on

93

a D8 Advance X-ray Diffractometer (Bruker AXS, Ltd., Germany) to analyze the sample crystal

94

structure. Fourier transform infrared (FT-IR) spectra was recorded on Nicolet Nexus 470 Fourier

95

transform infrared spectrophotometer (Thermo Electron Co., U.S.A.) using KBr pellet pressing

96

method to confirm the successful chemical modification of UCNPs. UV-2450 spectrophotometer

97

(Shimadzu, Japan) was used to measure the UV-vis absorption spectra of GNPs and verify the

98

aptamer modification. Upconversion fluorescence was tested on the assembled upconversion

99

fluorescence spectrophotometer, it assembly diagram can be found in Supplementary Information,

100

Fig. S1. The main components are 980 nm near infrared laser, SAC universal sample chamber,

101

Omni-λ monochromator, PMTH-S1 side-on photomultiplier tube, DCS103 data collector and

102

H1800V high voltage power supply. The zeta potential was measured using Malvern zetasizer Nano

103

ZS90 (Malvern Instruments Ltd., U.K.).

104

2.3 Development of biosensor

105 106

The chemical route followed to obtain the DNA modified upconversion nanoparticles and complementary DNA modified GNPs is given in Supplementary Information, Fig. S2 and Fig. S3.


Page 6 of 29

Page 7 of 29

107


2.3.1 Synthesis of aptamers-modified UCNPs

108

The procedure for preparation of oleic acid-capped upconversion nanoparticles (OA-UCNPs) was

109

performed based on previous research28 with some modifications. First, 0.2367 g of yttrium(III)

110

chloride hexahydrate, 0.0775 g of ytterbium(III) chloride hexahydrate and 0.0066 g of holmium (III)

111

chloride hexahydrate were weighed accurately and dispersed in 12 mL of methanol. Add this solution

112

into the 250 mL three-neck round-bottom flask whic contains 18 mL of oleic acid and 42 mL of

113

1-octadecene. A reflux condenser pipe whose lower end was fixedly arranged on the right above the

114

flask, was fixedly connected with the T-connector. The other two outlets of T-connector was

115

connected with inlet argon pipe and balloon. The other two necks of flask were attached to gas outlet

116

pipe and temperature rod, respectively. Put the flask on the magnetic heated stirrer. In the argon

117

protection environment, heat the above solution to 160 ºC under magnetic stirring and keep for 30

118

min, cooling naturally to 50 ºC. Then, turn off the argon intake valve and remove the reflux

119

condenser pipe from flask. Add 10 mL of methanol solution which involved 0.1482 g of NH4F and

120

0.1 g of NaOH into the above flask drop by drop. Next, heat up to 70 ºC and maintained it long

121

enough to promote full and evaporation of methanol. Then, re-attach the reflux condenser pipe on the

122

flask, turn on the argon intake valve and raise the heating temperature to 300 ºC, stirring constantly.

123

During this period, slow airflow velocity and inspect whether the bracket for the condensate pipe is

124

reliable from time to time. After the reaction, the solution appeared as transparent straw yellow.

125

Close the heating switch and allow the solution to cool down naturally to room temperature. The

126

nanoparticles were separated from the final solution by centrifugation and washed with the mixture

127

of cyclohexane and ethanol (v: v = 2: 1). Repeat three times. Dry the final product overnight in

128

vacuum oven at 55 ºC.



Page 8 of 29

129

The Carboxyl modification of UCNPs was carried out using the sequential procedure, first coat the

130

SiO2, second modify the amino groups and finally graft the carboxyl groups. The detail steps are

131

described as following. First, suspend 0.2 g of OA-UCNPs in 60 mL of ethanol, treat with ultrasonic

132

wave for 30 min to completely dissolve the power. Then, add 2.5 mL of 25% ammonia and 20 mL of

133

distilled water into the solution, seal off the flask and heat it to 70 ºC with stirring vigorously. After

134

keeping the temperature for 15 min, 200 µL of TEOS was added dropwise and the mixture was

135

stirred for another 6 h. Next, add 200 µL of APTES dropwise, keep stirring for 3 h, and cool slowly

136

to room temperature. The product was separated by centrifugation at 8000 rpm for 8 minutes, and

137

washed with ethanol. Repeat this process for three times, obtain amino-modified upconversion

138

nanoparticles (NH2-UCNPs). The resulting precipitate was dispersed in 15 mL methylbenzene, then

139

5 mL 0.16 M succinic anhydride in methylbenzene was added into above solution. Under the

140

protection of nitrogen, heating above solution to 80 ºC and continue to stir for a further 12 h. After

141

cool to room temperature, the production was separated by centrifugation (11000 rpm, 15 min),

142

washed with deionized water for three times. Finally, the precipitate (COOH-UCNPs) was added in a

143

vacuum drying oven and then dry it at 60 ºC for 4 h. 29

144

The procedure for the aptamer-functionalized UCNPs was adapted from a reported literature

145

First, 5 mg of carboxyl-modified UCNPs and 4 mg EDC were dispersed in 5 mL ultrapure water. Stir

146

for 15 min at room temperature to activating the catboxylic groups on the surface of UCNPs. Next, a

147

NHS solution in PBS buffer (10 mM, pH=7.4) was added into above solution, stir for a further 5 min.

148

Then, 700 µL 10 µM long-strand aptamer was added. As solution was stirred continuously,

149

carbodiimide coupling reaction took place, making the long-stranded aptamers with -NH2 covalently

150

attached to the COOH-UCNPs. After reaction for 12 h, the aptamers-modified UCNPs were purified


.

Page 9 of 29


151

by centrifuged at 12000 rpm for 10 min and washed with PBS buffer for three times. The final

152

production was re-dispersed in 10 mL PBS buffer and stored at 4 ºC.

153

2.3.2 Synthesis of aptamers-modified GNPs

154

GNPs were synthesized with citrate reduction method. First, all glass vessels used were cleaned in

155

aqua regia and washed under running ultrapure water. Then, the 250 mL round-bottom flask

156

contained 50 mL 1 mmol/L HAuCl4·4H2O solution was equipped with a reflux condenser and

157

incubated in the magnetic heated stirrer. Heat, stirring, until the solution boiled, add 5 mL 38.8

158

mmol/L trisodium citrate solution quickly. At this point, the solution changed from pale yellow color

159

to wine red. Allow it to boil for 30 min, cut down the power and leave the solution to cool naturally.

160

Pour the solution into the amber laboratory bottle and stored at 4 ºC. Transfer 5 mL of the prepared

161

GNPs solution to 10 mL centrifuge tube and add into 960 µL of 10 µM sulfhydryl modified

162

short-stranded aptamers. After 16 h incubation at 50 ºC, adjust the pH and salt concentration of

163

solution by introducing necessary salts, making the primary solution contain 0.1 M of NaCl and be at

164

pH 7.4. Then, keep the mixed solution at 50 ºC for a further 40 h. To purify the aptamer modified

165

GNPs, the above solution were centrifuged at 12000 rpm for 20 min, and the precipitate was washed

166

with PBS buffer for three times. The final wine red pellet was re-suspended in 5 mL PBS buffer and

167

stored at 4 ºC. Based on the Beer's law, the concentration of aptamers-modified GNPs was estimated

168

to be 200 nM, which was calculated by using their extinction coefficient of 3.6×108 cm-1M-1 at 528

169

nm and diameter of about 15 nm 30.

170

2. 4 Detection of Hg2+ by UCNPs -aptamers-GNPs biosensors

171

The upconversion fluorescence spectrophotometer was preheated for 30 min to obtain stable



172

signals before testing. The process of detecting Hg2+ is as follows: first, 5 mg/L aptamers-modified

173

UCNPs was mixed with aptamers-modified GNPs and hybridized for half an hour. Then, various

174

amounts of Hg2+ or other test samples were added into the above mixtures. After 0.5 h incubation at

175

room temperature, the mixture was measured using the assembled upconversion fluorescence

176

spectrofluorometer.

177

2. 5 Bio-detection of Hg2+ in spiked-in samples

178

For demonstrating the viability of this method, Hg2+ in tap water and milk was analyzed by the

179

constructed turn-on sensor. Typically, different concentrations of the standard solution of Hg2+ were

180

spiked into the pretreated tap water and milk and quantitatively detected by the developed method.

181

The pretreatment method of tap water and milk are as follow. Tap water was centrifuged and filtered

182

through a 0.22 µm membrane. Milk samples were purchased from a local supermarket. 20 mL of

183

milk was added to 4 mL of CHCl3 and 4 mL of 10% Cl3CCOOH solution. Then the mixture was

184

vortexed for 10 min on lab-dancer and centrifuged to remove proteins in milk. Subsequently, the

185

supernate was filtered through a 0.22 µM membrane to remove lipids. The gained filtrate was stored

186

for further use.

187

3. RESULTS AND DISCUSSION

188

3.1. Synthesis and surface functionalization of UCNPs and GNPs

189

TEM, XRD, FT-IR, UV-vis and zeta potential test were used to characterize the shape, size,

190

crystal form and surface modification of the nanoparticles in this study. As seen in Fig. 1A, the

191

morphology of OA-UCNPs dispersed in the hexane are highly monodisperse hexagonal polyhedra

192

with a mean diameter of 50 ± 3 nm. By X-ray diffraction method, crystal structure and matter phase


Page 10 of 29

Page 11 of 29


193

were obtain. It can be seen in Fig.1E, the XRD patterns of UCNPs shows pronounced peaks,

194

indicating the high crystallinity of the synthesized material. In order to improve the water solubility

195

of UCNPs to embroad their practical application, an efficient approach to modifying the surface of

196

UCNPs was presented. As illustrated in Fig. S2, the whole procedure consists of three steps: first,

197

coating silica shell on the UCNPs via the reverse microemulsion method; and then, modifying amine

198

groups by adding APTES; finally, grafting carboxylic groups onto the amino groups through ring

199

opening reaction of succinic anhydride. The inset in Fig. 1A depicts the TEM image of silica-coating

200

UCNPs. The shell thickness is about 3 nm, and the whole shell is integrated and uniform. The silica

201

shell could offer good hydrophilicity to UCNPs and serve as a platform for further modification. The

202

surface structure of the UCNPs after each modification step were confirmed by FT-IR spectroscopy.

203

As presented in Fig. 1F, the peak at 3400 cm−1 is corresponding to the stretching vibration of –OH

204

groups, peaks at 2930 cm−1 and 2853 cm−1 are ascribed to the asymmetric and symmetric stretching

205

vibrations of the methylene group (–CH2–), peaks at 1567 cm−1 and 1418 cm−1 are the characteristic

206

bands of asymmetric and symmetric stretching vibrations of the –COOH group (curve a), confirming

207

the existence of oleic acid on the surface of UCNPs. After modifying nanoparticles surface,

208

characteristic peaks at 1625 and 1068 cm−1 (curve b) appeared, which are of the stretching and

209

bending vibration of –NH2 groups and the stretching vibration of Si–O bond, validating successful

210

coating of SiO2 and modification of –NH2 groups (named NH2-UCNPs). After grafting –COOH by

211

reacting with succinic anhydride (named COOH-UCNPs), a new band of carbonyl groups at 1732

212

cm−1 appeared in the FT-IR spectrum, and meanwhile, the band at 3400 cm−1 disappeared (curve c).

213

This is because the successful graft of carboxylic groups onto amino groups and transform of

214

hydroxyl groups into carboxylic groups. Additionally, the surface potentials (ζ) of nanoparticles are



215

measured by zeta potential analyzer (Fig. 1I). Water-soluble NH2-UCNPs are positively-charged with

216

zeta potential of 22.3 ± 2.48 mV. After carboxyl modification, the zeta potential of UCNPs changes

217

into -26.7 ± 3.56 mV, reveals that the -COOH groups have been successfully grafted onto UCNPs.

218

The zeta potential of UCNPs-aptamers positively shift to -14.6 ± 2.14 mV, which is because of the

219

positive charges of amino-modified long-stranded aptamers. Besides, the synthesized GNPs show

220

zeta potential of -27.6 ± 3.52 mV, attributed to the citrate stabilizing agent in the surface. Upon

221

conjugation of the –SH-modified short-stranded aptamers, zeta potential of GNPs negatively shift to

222

-32.2 ± 3.76 mV, suggesting that the short-stranded aptamers modified GNPs are more stable in

223

water solution than the GNPs. Fig. 1B showes the TEM image of GNPs. GNPs have an average

224

diameter of 15 nm, and show good water solubility and dispersibility. The aptamers modification of

225

UCNPs and GNPs were verified from the results of UV-vis absorption spectroscopy (Fig. 1G). After

226

aptamers modification, the absorbance spectra of UCNPs and GNPs solution exhibit new peak at 260

227

nm, confirming the formation of long-stranded aptamers modified UCNPs and short-stranded

228

aptamers modified GNPs.

[Here for Fig. 1]

229

230

3.2. Sensing scheme

231

A schematic of the turn-on fluorescent sensor for Hg2+ based on the FRET pair of UCNPs and

232

GNPs was shown in Fig. 2. Two single-stranded aptamers (long-stranded aptamers and

233

short-stranded aptamers) were modified in UCNPs and GNPs, respectively. In the absence of Hg2+,

234

the total 10 complementary base pairs of short-stranded aptamers were hybridized with the

235

corresponding bases of long-stranded aptamers, bringing GNPs to the proximity of UCNPs, leading


Page 12 of 29

Page 13 of 29


236

to green fluorescence quenching of UCNPs. This is because the overlap between the absorption

237

spectrum of GNPs and the fluorescence spectrum of UCNPs in 545 nm (inset a in Fig. 2) that cause

238

FRET between them. When Hg2+ was introduced, long-strand aptamer on the surface of UCNPs

239

folded into hairpin structure, which is attributed to the reason that T-T mispairs in long-strand

240

aptamer can selectively capture Hg2+ ions and form T-Hg2+-T base pairs (inset b in Fig. 2). In this

241

case, only six base pairs between long-stranded aptamer and short-stranded aptamer left. Besides,

242

Since the van der Waals radius of mercury is ∼1.44 Å while the base pair spacing in DNA duplex is

243

∼3.4 Å, suggesting the T-Hg2+-T pair is more stable than the Watson-Crick A-T pair31. Therefore,

244

GNPs modified with short-stranded aptamer release from UCNPs, resulting in the disappearance of

245

FRET and the recover of upconversion fluorescence. The developed UCNPs-aptamers-GNPs

246

detection system was characterized by TEM. In absence of Hg2+, many GNPs attaches to UCNPs,

247

which can be seen in Fig. 1C. In presence of Hg2+, the number of GNPs attached in UCNPs

248

significantly decreased (Fig. 1D), successfully indicated the sensing principle of the detection.

249

[Here for Fig. 2]

250

To investigate the process of fluorescence resonance energy transfer, as shown in Fig. 3A, the

251

strong fluorescence of the UCNPs was gradually quenched with the increase of GNPs concentrations.

252

This fluorescence quenching of UCNPs is attributed to the FRET from UCNPs to GNPs via the

253

short-stranded aptamers modified on the GNPs surface binding the long-stranded aptamers on the

254

UCNPs surface. The fluorescence quenching was defined by the following equation: E = (Fi－F0)/F0,

255

in which Fi and F0 refer to the upconversion fluorescence intensities of the UCNPs in the presence

256

and absence of the GNPs. As seen in Fig. 3B, the quenching efficiency increased with the

257

concentration of GNPs. When the concentration of GNPs reached 170 nM, the quenching efficiency ACS Paragon Plus Environment


258

tended to grow sluggishly. Therefore, 170 nM was selected as the final concentration of GNPs for the

259

subsequent experiments. In this case, the fluorescence quenching efficiency reaches 85.4%, which

260

offers favorable conditions for sensitive detection of target.

261

[Here for Fig. 3]

262

3.3. Optimization of detection conditions

263

To obtain high performance of the developed sensor, the hybridization time of aptamers-modified

264

UCNPs and aptamers-modified GNPs, and the reaction time after adding Hg2+ were optimized in this

265

study. The fluorescence changes of FRET system in the presence of 5 mg/L aptamers-modified

266

UCNPs and 170 nM aptamers-modified UCNPs for a fixed time interval of 3 min are shown in Fig.

267

3C. The fluorescence intensity decreased gradually over time. When the hybridization time reached

268

18 min, the fluorescence intensity stopped decrease due to the almost complete hybridization of

269

aptamers-modified UCNPs and aptamers-modified GNPs. To ensure the fully hybridization, an

270

optimum 21 min hybridization time was applied in the subsequent experiments. The latter part of Fig.

271

3C shown the changing trend of fluorescence intensity with the reaction time after adding Hg2+. The

272

fluorescence intensity was found to be increased gradually within the first 9 min, however, no

273

prominent increase was observed after 9 min because almost all the Hg2+ had combined with

274

thymine in the long-strand aptamers to make the FRET disappear. Based on this study, 9 min reaction

275

time was used in the further experiments. All the experiment were performed at room temperature

276

(25−28℃).

277

3.4. Fluorescence detection of Hg2+ based on UCNPs-aptamers-GNPs FRET sensor

278

To demonstrate the performance of the developed FRET sensor for Hg2+ detection, the detection


Page 14 of 29

Page 15 of 29


279

range and LOD were investigated. Fig. 4A shows UC fluorescence spectra against the concentrations

280

of Hg2+ in the homogeneous assay. As the concentration of Hg2+ increased, the fluorescence intensity

281

at 545 nm increased gradually. The plot of fluorescence intensity against the concentrations of Hg2+

282

is shown in Fig. 4B. A linear correlation between the fluorescence intensities and Hg2+ concentrations

283

is obtained in low concentration regions (0.2-20 µM), which can be fitted as y = 83.351x+414.86,

284

with R2 = 0.9947. The LOD can be calculated by 3S0/S, where S0 represents the relative standard

285

deviation of the measured value of nine blank samples, and S is the slope of the calibration curve (S0

286

= 1.69, S = 83.351). In this study, a value of 60 nM was obtained, which is lower than the safe level

287

for food (10 µM). Thus, this sensor can detect Hg2+ quantitatively at low concentration.

288

289

[Here for Fig. 4]

3.5. Selectivity and anti-interfering of UCNPs-aptamers-GNPs FRET system

290

To explore the selectivity of the developed system, several metal ions (K+, Na+, Mn2+, Ba2+, Cu2+,

291

Ca2+, Cd2+, Mg2+, Zn2+, Pb2+ and Fe3+) were examined. As shown in Fig. 5, only Hg2+ resulted in a

292

high response, while none of these metal ions caused fluorescence “turn-on” even in 10 times

293

concentration, demonstrating the high selectivity of the FRET system. Besides, anti-interfering test

294

was investigated. The results in Fig. 5 shows that, fluorescence intensity restored even if other metal

295

ions existed. Besides, nearly no fluorescence intensity difference was observed when detecting 10

296

µM of Hg2+ by the FRET sensor whether other metal ions existed. Therefore, this detection system is

297

rather selective and other metal ions do not interfere with the Hg2+ detection, indicating that the

298

developed system can be used to detect Hg2+ in real samples.

299

[Here for Fig. 5]



300

3.6. Hg2+ determination in real samples

301

To test whether the developed sensor could be further applied in real samples, the spiked known

302

amount of Hg2+ into tap water and pre-treated milk samples were detected and investigated. The

303

prepared tap water and milk were spiked into Hg2+ on three different level of concentrations.

304

Through recording the corresponding fluorescence intensity values and substituted them in the linear

305

formula, we calculated the test values and further obtained the recoveries. Meanwhile, intra- and

306

inter-assay coefficients of variation (CV) were measured to analysis the reliability of the sensor. As

307

shown in Table 1, the quantitative detection of spiked samples with three different concentration of

308

Hg2+ was achieved with recoveries in the range of 95.18% to 108.22%, intra-assay CV in range of

309

2.03% to 7.07%. and inter-assay CV in the range of 5.31% to 8.57%. These results revealed the

310

feasibility of the developed method for Hg2+ detection in real samples. Furthermore, a comparison of

311

the developed method in this work with other published fluorescence methods for the detection of

312

Hg2+ ions is presented in Table S1. Compared with DAM and Eu3+ hybrid carbon dots32, 33, the

313

developed UCNPs-aptamers-GNPs exhibited higher sensitivity for Hg2+ ions detection. Even though

314

the sensitivity of the method for Hg2+ ion is not comparable with that of other methods in the

315

literature34-39, but it exhibits simplicity, accuracy and rapidity for detection of Hg2+ ions. Meanwhile,

316

its its excellent water-soluble performance can make it more extensive applications in practice.

[Here for Table 1]

317

318

ASSOCIATED CONTENT

319

Supporting Information

320

Assembly diagram of the instrument can be found in Fig. S1. The chemical route followed to


Page 16 of 29

Page 17 of 29


321

obtain the DNA modified upconversion nanoparticles and complementary DNA modified GNPs is

322

given in Fig. S2 and Fig. S3. A comparison of the developed method in this work with other

323

published fluorescence methods for the detection of Hg2+ ions is presented in Table S1.

324

AUTHOR INFORMATION

325

Corresponding Author

326

*Telephone.: +86-511-88790318.

327

Fax: +86-511-88780201.

328

E-mail: [email protected] (Q.S.Chen).

329

Funding This work has been financially supported by the National Natural Science Foundation of China

330 331

(31772063).

332

Notes

The authors declare no competing financial interest.

333

334

ACKNOWLEDGEMENT

The authors are thankful to the authorities of Jiangsu University for providing research facilities to

335 336

carry out this work.

337

REFERENCES

338

1.

339

impacts. Journal of Hazardous Materials 2016, 306, 376-385.

Kim, K.-H.; Kabir, E.; Jahan, S. A., A review on the distribution of Hg in the environment and its human health



340

2.

341

and mercury ions in the aqueous solution. Sensors and Actuators B: Chemical 2018, 257, 728-733.

342

3.

343

catappa leaves as antibacterial agent and colorimetric mercury sensor. Materials Letters 2017, 207, 66-71.

344

4.

345

Hg2+ ions. Sensors and Actuators B: Chemical 2017, 249, 217-228.

346

5.

347

pyrido[1,2-a]benzimidazole-rhodamine system. Sensors and Actuators B: Chemical 2017, 251, 410-415.

348

6.

349

chemosensor for ratiometric detection Hg2+ in actual aqueous samples. Journal of Luminescence 2017, 188, 135-140.

350

7.

351

copper and biothiols based on a benzothiazole derivative. Spectrochimica Acta Part A: Molecular and Biomolecular

352

Spectroscopy 2018, 191, 427-434.

353

8.

354

binding of Hg2+ ions by three related receptors. Inorganica Chimica Acta 2017, 462, 152-157.

355

9.

356

bioimaging of Hg2+ and Cu2+. Analytica Chimica Acta 2017, 954, 97-104.

357

10. Singh, A.; Singh, A.; Singh, N.; Jang, D. O., Selective detection of Hg(II) with benzothiazole-based fluorescent

358

organic cation and the resultant complex as a ratiometric sensor for bromide in water. Tetrahedron 2016, 72,

359

3535-3541.

360

11. Feng, L.; Deng, Y.; Wang, X.; Liu, M., Polymer fluorescent probe for Hg(II) with thiophene, benzothiazole and

361

quinoline groups. Sensors and Actuators B: Chemical 2017, 245, 441-447.

362

12. Vasimalai, N.; John, S. A., Ultrasensitive and selective spectrofluorimetric determination of Hg(II) using a

363

dimercaptothiadiazole fluorophore. Journal of Luminescence 2011, 131, 2636-2641.

364

13. Chang, I. J.; Hwang, K. S.; Chang, S.-K., Selective Hg2+ signaling via dithiane to aldehyde conversion of an ESIPT

365

fluorophore. Dyes and Pigments 2017, 137, 69-74.

366

14. Castillo, J.; Chirinos, J.; Gutiérrez, H.; La Cruz, M., Surface plasmon resonance sensor based on golden

367

nanoparticles and cold vapour generation technique for the detection of mercury in aqueous samples. Optics & Laser

368

Technology 2017, 94, 34-39.

369

15. Chang, C.-C.; Lin, S.; Wei, S.-C.; Chen, C.-Y.; Lin, C.-W., An amplified surface plasmon resonance “turn-on” sensor for

370

mercury ion using gold nanoparticles. Biosensors and Bioelectronics 2011, 30, 235-240.

Kim, S. K.; Gupta, M.; Lee, H.-i., A recyclable polymeric film for the consecutive colorimetric detection of cysteine

Devadiga, A.; Vidya Shetty, K.; Saidutta, M. B., Highly stable silver nanoparticles synthesized using Terminalia

Ozdemir, M., A fast-response, highly selective, chromogenic and fluorescent chemosensor for the detection of

Ge, Y.; Liu, A.; Ji, R.; Shen, S.; Cao, X., Detection of Hg2+ by a FRET ratiometric fluorescent probe based on a novel

Xu, N.-Z.; Liu, M.-M.; Ye, M.-A.; Yao, Y.-W.; Zhou, Y.; Wu, G.-Z.; Yao, C., A Rhodamine-naphthalimide conjugated

Shen, Y.; Zhang, X.; Zhang, C.; Zhang, Y.; Jin, J.; Li, H., A simple fluorescent probe for the fast sequential detection of

Dhaka, G.; Singh, J.; Kaur, N., Benzothiazole based chemosensors having appended amino group(s): Selective

Gu, B.; Huang, L.; Su, W.; Duan, X.; Li, H.; Yao, S., A benzothiazole-based fluorescent probe for distinguishing and


Page 18 of 29

Page 19 of 29


371

16. Khodaveisi, J.; Shabani, A. M. H.; Dadfarnia, S.; Moghadam, M. R.; Hormozi-Nezhad, M. R., Development of a novel

372

method for determination of mercury based on its inhibitory effect on horseradish peroxidase activity followed by

373

monitoring the surface plasmon resonance peak of gold nanoparticles. Spectrochimica Acta Part A: Molecular and

374

Biomolecular Spectroscopy 2016, 153, 709-713.

375

17. Rithesh Raj, D.; Prasanth, S.; Vineeshkumar, T. V.; Sudarsanakumar, C., Surface Plasmon Resonance based fiber

376

optic sensor for mercury detection using gold nanoparticles PVA hybrid. Optics Communications 2016, 367, 102-107.

377

18. Sadrolhosseini, A. R.; Naseri, M.; Rashid, S. A., Polypyrrole-chitosan/nickel-ferrite nanoparticle composite layer for

378

detecting heavy metal ions using surface plasmon resonance technique. Optics & Laser Technology 2017, 93, 216-223.

379

19. Li, F.; Wang, J.; Lai, Y.; Wu, C.; Sun, S.; He, Y.; Ma, H., Ultrasensitive and selective detection of copper (II) and

380

mercury (II) ions by dye-coded silver nanoparticle-based SERS probes. Biosensors and Bioelectronics 2013, 39, 82-87.

381

20. Song, C.; Yang, B.; Zhu, Y.; Yang, Y.; Wang, L., Ultrasensitive sliver nanorods array SERS sensor for mercury ions.

382

Biosensors and Bioelectronics 2017, 87, 59-65.

383

21. Xu, L.; Yin, H.; Ma, W.; Kuang, H.; Wang, L.; Xu, C., Ultrasensitive SERS detection of mercury based on the

384

assembled gold nanochains. Biosensors and Bioelectronics 2015, 67, 472-476.

385

22. Xu, W.; Chen, X.; Song, H., Upconversion manipulation by local electromagnetic field. Nano Today 2017, 17, 54-78.

386

23. Liu, Y.; Ouyang, Q.; Li, H.; Zhang, Z.; Chen, Q., Development of an Inner Filter Effects-Based Upconversion

387

Nanoparticles–Curcumin Nanosystem for the Sensitive Sensing of Fluoride Ion. ACS Applied Materials & Interfaces 2017,

388

9, 18314-18321.

389

24. Chen, Q.; Hu, W.; Sun, C.; Li, H.; Ouyang, Q., Synthesis of improved upconversion nanoparticles as ultrasensitive

390

fluorescence probe for mycotoxins. Analytica Chimica Acta 2016, 938, 137-145.

391

25. Hu, W.; Chen, Q.; Li, H.; Ouyang, Q.; Zhao, J., Fabricating a novel label-free aptasensor for acetamiprid by

392

fluorescence resonance energy transfer between NH2-NaYF4: Yb, Ho@SiO2 and Au nanoparticles. Biosensors and

393

Bioelectronics 2016, 80, 398-404.

394

26. Wang, S.; Han, M.-Y.; Huang, D., Nitric Oxide Switches on the Photoluminescence of Molecularly Engineered

395

Quantum Dots. Journal of the American Chemical Society 2009, 131, 11692-11694.

396

27. Zhang, S.; Sun, M.; Yan, Y.; Yu, H.; Yu, T.; Jiang, H.; Zhang, K.; Wang, S., A turn-on fluorescence probe for the

397

selective and sensitive detection of fluoride ions. Analytical and Bioanalytical Chemistry 2017, 409, 2075-2081.

398

28. Chen, Q.; Hu, W.; Sun, C.; Li, H.; Ouyang, Q., Synthesis of improved upconversion nanoparticles as ultrasensitive

399

fluorescence probe for mycotoxins. Analytica Chimica Acta 2016, 938, 137-145.

400

29. Xie, X.; Li, F.; Zhang, H.; Lu, Y.; Lian, S.; Lin, H.; Gao, Y.; Jia, L., EpCAM aptamer-functionalized mesoporous silica

401

nanoparticles for efficient colon cancer cell-targeted drug delivery. European Journal of Pharmaceutical Sciences 2016,



402

83, 28-35.

403

30. Griffin, J.; Singh, A. K.; Senapati, D.; Lee, E.; Gaylor, K.; Jones-Boone, J.; Ray, P. C., Sequence-Specific HCV RNA

404

Quantification Using the Size-Dependent Nonlinear Optical Properties of Gold Nanoparticles. Small 2009, 5, 839-845.

405

31. Miyake, Y.; Togashi, H.; Tashiro, M.; Yamaguchi, H.; Oda, S.; Kudo, M.; Tanaka, Y.; Kondo, Y.; Sawa, R.; Fujimoto, T.;

406

Machinami, T.; Ono, A., MercuryII-Mediated Formation of Thymine−HgII−Thymine Base Pairs in DNA Duplexes. Journal

407

of the American Chemical Society 2006, 128, 2172-2173.

408

32. Zhou, S.; Zhou, Z.-Q.; Zhao, X.-X.; Xiao, Y.-H.; Xi, G.; Liu, J.-T.; Zhao, B.-X., A dansyl based fluorescence chemosensor

409

for Hg2+ and its application in the complicated environment samples. Spectrochimica Acta Part A: Molecular and

410

Biomolecular Spectroscopy 2015, 148, 348-354.

411

33. Venkatesan, P.; Thirumalivasan, N.; Wu, S.-P., A rhodamine-based chemosensor with diphenylselenium for highly

412

selective fluorescence turn-on detection of Hg2+ in vitro and in vivo. RSC Advances 2017, 7, 21733-21739.

413

34. Desai, M. L.; Jha, S.; Basu, H.; Singhal, R. K.; Sharma, P. K.; Kailasa, S. K., Microwave-assisted synthesis of

414

water-soluble Eu3+ hybrid carbon dots with enhanced fluorescence for the sensing of Hg2+ ions and imaging of fungal

415

cells. New Journal of Chemistry 2018.

416

35. Luo, T.; Zhang, S.; Wang, Y.; Wang, M.; Liao, M.; Kou, X., Glutathione-stabilized Cu nanocluster-based fluorescent

417

probe for sensitive and selective detection of Hg2+ in water. Luminescence 2017, 32, 1092-1099.

418

36. Wang, G.; Lu, Y.; Yan, C.; Lu, Y., DNA-functionalization gold nanoparticles based fluorescence sensor for sensitive

419

detection of Hg2+ in aqueous solution. Sensors and Actuators B: Chemical 2015, 211, 1-6.

420

37. Ding, H.; Zheng, C.; Li, B.; Liu, G.; Pu, S.; Jia, D.; Zhou, Y., A rhodamine-based sensor for Hg2+ and resultant complex

421

as a fluorescence sensor for I. RSC Advances 2016, 6, 80723-80728.

422

38. Chen, B.; Ma, J.; Yang, T.; Chen, L.; Gao, P. F.; Huang, C. Z., A portable RGB sensing gadget for sensitive detection of

423

Hg2+ using cysteamine-capped QDs as fluorescence probe. Biosensors and Bioelectronics 2017, 98, 36-40.

424

39. Ma, X.; Song, F.; Wang, L.; Cheng, Y.; Zhu, C., Polymer‐based colorimetric and “turn off” fluorescence sensor

425

incorporating benzo[2,1,3]thiadiazole moiety for Hg2+ Detection. Journal of Polymer Science Part A Polymer Chemistry

426

2015, 50, 517-522.

427


Page 20 of 29

Page 21 of 29


428

Figure Captions

429

In this work, five figures were constructed for further demonstration with brief descriptions as

430

follows:

431

Fig. 1. (A) TEM image of UCNPs (inset: TEM image of UCNPs@SiO2). (B) TEM image of GNPs.

432

(C) TEM image of UCNPs-aptamers-GNPs in absence of Hg2+. (D) TEM image of

433

UCNPs-aptamers-GNPs in presence of Hg2+. (E) XRD of UCNPs. (F) FT-IR spectra of (a) UCNPs,

434

(b) amino-modified UCNPs, (c) carboxyl-modified UCNPs. (G) a. UV-vis spectra of the UCNPs

435

before and after aptamers modification; b. UV-vis spectra of the GNPs before and after aptamers

436

modification. (H) Zeta potential of UCNPs-NH2, UCNPs-COOH, UCNPs-aptamers, GNPs and

437

GNPs-aptamers.

438

Fig. 2. Schematic Description of the UCNPs-aptamers-GNPs FRET sensor for Hg2+.

439

Fig. 3. (A) Upconversion fluorescence spectra of the 100 µL 5 mg/L UCNPs after addition of equal

440

volume of GNPs with different concentrations (0, 20, 30, 40, 50, 60, 70, 90, 100, 110, 120, 140, 170,

441

200 nM). (B) Plot of fluorescence quenching efficiency versus GNPs concentration. (C)

442

Optimization experiments of hybridization and reaction time (20 µM was selected as the Hg2+

443

concentration to determine the optimum hybridization and reaction time).

444

Fig. 4. (A) Upconversion fluorescence spectra of the UCNPs-aptamers-GNPs in the presence of

445

different concentrations of Hg2+. (B) Plot of upconversion fluorescence intensity at 545 nm versus

446

Hg2+ concentrations (0.2, 0.4, 0.8, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 µM).

447

Fig. 5. Selectivity and anti-interfering of the developed UCNPs-aptamers-GNPs FRET sensor. The



448

concentrations of Hg2+ and other metal ions are 10 µM and 100 µM. Every data point was the mean

449

of three measurements. The error bars are the standard deviation.

450


Page 22 of 29

Page 23 of 29

451


Table 1. Determination of Hg2+ in real samples using the developed method Intra-assay

Inter-assay

Sample Found±SD

Recovery

CVs

Found±SD

Recovery

CVs

(µM)

(%)

(%)

(µM)

(%)

(%)

0.2

0.216 ± 0.015

108.22%

7.07

0.196 ± 0.017

98.18

8.57

2

2.08 ± 0.11

104.08%

5.24

1.93 ± 0.14

96.61

7.19

20

20.90 ± 1.01

104.51%

4.81

20.62 ± 1.73

103.1

8.37

0.2

0.190 ± 0.009

95.18

4.76

0.199 ± 0.016

99.72

8.12

2

1.97 ± 0.11

98.45

5.96

2.07 ± 0.11

103.77

5.31

20

19.91 ± 0.40

99.55

2.03

19.92 ± 1.07

99.60

5.39

(µM)

Tap water

Milk

452

SD: standard deviation; CVs: coefficient of variations.

453



454 455

TOC Graphic. Schematic Description of the UCNPs-aptamers-GNPs FRET sensor for Hg2+

456 457


Page 24 of 29

Page 25 of 29


Fig. 1. (A) TEM image of UCNPs (inset: TEM image of UCNPs@SiO2). (B) TEM image of GNPs. (C) TEM image of UCNPs-aptamers-GNPs in absence of Hg2+. (D) TEM image of UCNPs-aptamers-GNPs in presence of Hg2+. (E) XRD of UCNPs. (F) FT-IR spectra of (a) UCNPs, (b) amino-modified UCNPs, (c) carboxyl-modified UCNPs. (G) a. UV-vis spectra of the UCNPs before and after aptamers modification; b. UV-vis spectra of the GNPs before and after aptamers modification. (H) Zeta potential of UCNPs-NH2, UCNPs-COOH, UCNPs-aptamers, GNPs and GNPs-aptamers. 115x202mm (300 x 300 DPI)



Fig. 2. Schematic Description of the UCNPs-aptamers-GNPs FRET sensor for Hg2+. 289x157mm (300 x 300 DPI)


Page 26 of 29

Page 27 of 29


Fig. 3. (A) Upconversion fluorescence spectra of the 100 µL 5 mg/L UCNPs after addition of equal volume of GNPs with different concentrations (0, 20, 30, 40, 50, 60, 70, 90, 100, 110, 120, 140, 170, 200 nM). (B) Plot of fluorescence quenching efficiency versus GNPs concentration. (C) Optimization experiments of hybridization and reaction time (20 µM was selected as the Hg2+ concentration to determine the optimum hybridization and reaction time). 227x59mm (300 x 300 DPI)



Fig. 4. (A) Upconversion fluorescence spectra of the UCNPs-aptamers-GNPs in the presence of different concentrations of Hg2+. (B) Plot of upconversion fluorescence intensity at 545 nm versus Hg2+ concentrations (0.2, 0.4, 0.8, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 µM). 166x65mm (300 x 300 DPI)


Page 28 of 29

Page 29 of 29


Fig. 5. Selectivity of the developed UCNPs-aptamers-GNPs FRET sensor. The concentrations of Hg2+ and other metal ions are 10 µM and 100 µM. Every data point was the mean of three measurements. The error bars are the standard deviation. 75x59mm (300 x 300 DPI)


A Turn-on Fluoresence Sensor for Hg2+ in Food Based on FRET

Recommend Documents